Aqueous ifosfamide compositions for parenteral administration and a process for thier preparations

ABSTRACT

The present invention provides aqueous Ifosfamide compositions and a process for their preparation, in which the compositions have a reduced toxicity over and above the concomitant use of the uroprotective agent, Mesna. Aqueous Ifosfamide compositions can be prepared at concentrations as high has 1,1000 mg/ml.

[0001] This application claims priority to Indian provisionalapplication 785/mum/02 filed Dec. 2, 2002; and Indian application number______ (number awaited), filed Dec. 2, 2003; and PCT application, ______(number awaited), filed Dec. 2, 2003, all of which are hereinincorporated in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to aqueous Ifosfamide compositionscomprising 2-hydroxypropyl-β-cyclodextrin (referred to hereinafter as“HPBCD”), which are suitable for parenteral use. This inventionparticularly relates to stable, clear, aqueous, ready to use, as well asconcentrated, Ifosfamide compositions having reduced toxicity, as wellas methods for their preparation.

BACKGROUND OF THE INVENTION

[0003] Two main groups of drugs used in the treatment of malignantdisease are alkalyting agents and the antimetabolites. Ifosfamide is oneof the widely used antineoplastic drug belonging to the alkalytingagents group.

[0004] Ifosfamide is chemically3-(2-chloroethyl)-2-[(2-chloroethyl)amino]-tetrahydro-2H-1,3,2-oxazaphosphorin-2-oxideand is represented by the formula:

[0005] Ifosfamide is a white crystalline hygroscopic powder having a lowmelting point of 40° C. The powder has a water solubility of about 100mg/ml.

[0006] Ifosfamide is used in the treatment of a variety of solid tumoursincluding those of the cervix, endometrium, lung, ovary, testes andthymus as well as in sarcoma and in the treatment of Burkitts lymphoma.However, the treatment with Ifosfamide is associated with serious sideeffects such as haemorrhagic cystitis, myelosuppression, cardiacarrythmias, CNS disturbances, nephrotoxicity, haematological andgastro-intestinal reactions. The LD₅₀ in mouse on intravenousadministration has been reported to be 338 mg/kg body weight.Combination with the uroprotective agent, mesna, reduces the incidenceof hamorrhagic cystitis. Thus, mesna is normally administeredintravenously at a dose of 20% of the Ifosfamide dose at time zero (thetime of administration of Ifosfamide), and then at 4 and 8 hours.

[0007] Typically, Ifosfamide is given intravenously either by injectionor by infusion as a diluted solution containing less than 4% w/v ofIfosfamide. Ifosfamide is very susceptible to hydrolytic degradation andaccordingly prompt administration of such solutions is generallyrequired. Therefore, commercially it is predominately available in dryform and is supplied as sterile packaged dry powder for dissolution inwater for injection prior to administration. However, the low meltingpoint and the hygroscopic nature of Ifosfamide make it necessary to fillthe powder with great care accurately controlling both temperature andhumidity to achieve a sterile product. Further, prolonged storage of thedry powder also results in sintering and yellowing, which in turn leadsto a reduction in dissolution rate thereby increasing the time necessaryfor reconstitution.

[0008] To overcome difficulties associated with the thermal andhydrolytic susceptibility, lyophilization of the drug has beenattempted. However, the lyophilization process is quite time consumingand requires specialized equipment. Personnel exposure to the stronglycytotoxic Ifosfamide occurring during reconstitution of lyophilizedpowder is also very undesirable.

[0009] Hence, others have attempted to obtain a clear liquid Ifosfamidecompositions, suitable for parenteral administration, that are stableover a period of time

[0010] U.S. Pat. No. 4,952,575 discloses preparation of an ethanolicsolution of Ifosfamide containing 96% to 100% ethanol. Even though thedegradation of Ifosfamide has been shown to be minimal, use of solventsin such a high concentration leads to other problems such as volatility,handling during manufacturing, and miscibility with blood onadministration. As such, alcohol is pharmacologically active, which mayalso effect the person on administration of alcoholic solution ofIfosfamide.

[0011] U.S. Pat. No. 4,879,286 discloses a Cyclophosphamide formulatedin a ready-to-dilute solution. The solution has organic polyols, namelypropylene glycol and polyethylene glycol and their mixtures, as asolvent and also 0 to 50% water. The water may be partly replaced by 10to 30% of ethanol.

[0012] WO 02/02125 discloses a liquid pharmaceutical composition forparenteral administration comprising Ifosfamide, solvent and optionally,conventional pharmaceutical carriers and excipients. According to theapplication, the solvent comprises 35-75% lower alcohol and 25-65%polyol. While lower alcohol solvent is usually ethanol, the polyolsolvent is propylene glycol, glycerol and/or polyethylene glycol.

[0013] In both the U.S. Pat. No. 4,879,286 and WO 02/02125, theparenteral administration of larger amounts of polyols and alcohols leadto other problems like pain or irritation on injection, hemolysis,ototoxicity, cardiovascular effects, CNS effects and seizures. It mayalso lead to hyperosmolarity and lactic acidosis in patients with renalimpairment.

[0014] WO 99/18973 describes a stable, ready-to-use liquor of Ifosfamideusing Sodium chloride as a stabilizing agent. The invention alsodescribes 10-500 mg/ml Ifosfamide composition containing Urea, sodiumchloride and sodium dihydrogen phosphate. The compositions of theinvention are said to be stable but there is no mention about the safetyand toxicity of the composition. The higher concentration of urea in theformulation may lead to complications like hemolysis, irritation,phlebitis & thrombosis at the site of injection, and elevated bloodammonia & urea concentrations in patients with hepatic and renalfunction impairment.

[0015] WO 03/051297A2 describes a ready-to-use aqueous composition ofIfosfamide comprising 40-400 mM (10-100 mg/ml) of Ifosfamide in apharmaceutically acceptable buffer. The patent suggests the use ofbuffers preferably from the group of Na₂HPO₄ and NaH₂PO₄ and K₂HPO₄ andKH₂PO₄. There is no report on the toxicity of Ifosfamide compositionsdisclosed in this patent.

[0016] Thus, there remains a need for a stable, concentrated Ifosfamidesolution to facilitate handling during administration. In addition,there remains a need for Ifosfamide pharmaceutical compositions thatexhibit less toxicity than the currently available compositions. Thepresent invention meets this need.

SUMMARY OF THE INVENTION

[0017] The present invention provides aqueous compositions comprisingIfosfamide and 2-hydroxylpropyl-β-cyclodextrin. Preferably theIfosfamide is present at a concentration up to 1,100 mg/ml. Otherpreferred Ifosfamide concentration ranges include 1-200 mg/ml; 10-100mg/ml; 40-50 mg/ml; 200-500 mg/ml; 500-1,000 mg/ml; and greater than1,000 mg/ml.

[0018] In aqueous compositions of the present invention, the2-hydroxypropyl-β-cyclodextrin has a varying range of molar substitutionby hydroxy propyl groups, including 0.05-2; 0.3-1.5; and 0.5-1.2.

[0019] In aqueous compositions of the present invention, the molar ratioof Ifosfamide to 2-hydroxypropyl-β-cyclodextrin is preferably100:0.1-1:300; or 100:0.25-1:100; or 100:1-1:20; or 100:3.3-1:2.5.

[0020] The compositions of the present invention may further comprisepharmaceutically acceptable buffers, tonicity agents, preservatives,chelating agents, antioxidants, and/or anti-crystallizing agents.

[0021] Preferably the compositions of the present invention have a pHbetween 3.0-9.0 More preferably, between 5.0-8.0.

[0022] Suitable buffering agents include, but are not limited to, Sodiumdihydrogen phosphate, Disodium hydrogen phosphate, Dipotassium hydrogenphosphate, Potassium dihydrogen phosphate, Histidine HCl, SodiumHydroxide, Phosphoric acid, and Hydrochloric acid and mixtures thereof.A preferred buffering agent is a mixture of Sodium dihydrogen phosphateand Disodium hydrogen phosphate.

[0023] The present invention also provides process for the preparationof Ifosfamide compositions, whereby Ifosfamide is brought in intimatecontact with 2-hydroxypropyl-β-cyclodextrin in water to form aIfosfamide/2-hydroxyl-β-cyclodextrin solution. The Ifosfamide may bepresent in a concentration up to 1,100 mg/ml. Exemplary concentrationsof Ifosfamide include 1-200 mg/ml; 10-100 mg/ml; 40-50 mg/ml; 200-500mg/ml; 500-1,000 mg/ml; and greater than 1,000 mg/ml.

[0024] The 2-hydroxypropyl-β-cyclodextrin has a preferable molarsubstitution as described in the compositions of the present invention.

[0025] The molar ratio of Ifosfamide to 2-hydroxypropyl-β-cyclodextrinis preferably as described above in the compositions of the presentinvention.

[0026] In the process of the present invention, the addition of one ormore pharmaceutically acceptable buffers, tonicity agents,preservatives, chelating agents, antioxidants, or anti-crystallizingagents may occur while bringing in intimate contact the Ifosfamide withthe 2-hydroxylproplyl-β-cyclodextrin, or may be added to theIfosfamide/2-hydroxylproplyl-β-cyclodextrin solution.

[0027] The solution may be sterilized and is preferably sterilized byfiltering through a 0.2 μm filter. After sterilization, it is preferablyto place the solution into sterile containers followed by purging of theair in the headspace of the containers with an inert gas such asnitrogen.

[0028] One embodiment of the present invention includes an aqueousIfosfamide composition wherein 1 ml of the composition comprises 500 mgIfosfamide, and 400 mg of 2-hydroxylpropyl-β-cyclodextrin.

[0029] Another embodiment of the present invention provides an aqueousIfosfamide composition wherein 1 ml of the composition comprises 1000 mgIfosfamide, and 50 mg of 2-hydroxylpropyl-β-cyclodextrin.

[0030] Yet another embodiment of the present invention provides anaqueous Ifosfamide composition wherein 1 ml of the composition comprises50 mg Ifosfamide, and 100 mg of 2-hydroxylpropyl-β-cyclodextrin, and 0.3mg Sodium dihydrogen phosphate, and 0.5 mg Disodium hydrogen phosphate.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides an Ifosfamide composition that isstable, clear, aqueous, sterile and ready to use. Compositions of thepresent invention may be concentrated, and they exhibit a reducedtoxicity, as compared to currently available Ifosfamide compositions.Compositions of the present invention also have a reduced toxicity overand above the concomitant use of the uroprotective agent, Mesna.

[0032] The present invention provides an aqueous composition comprisingIfosfamide and 2-hydroxylpropyl-β-cyclodextrin (HPBCD) that provides fora reduced toxicity, as compared to conventional Ifosfamide formulations(i.e. formulations not having HPBCD), both in the presence and absenceof Mesna. Further, the present inventors have discovered that Ifosfamide(although a soluble drug) has an even greater solubility in HPBCD, whichallows for the manufacture of concentrated Ifosfamide compositions,heretofore believed unachievable. It was believed that the highestconcentration achievable in water was about 100 mg/ml. The presentinvention provides aqueous compositions of Ifosfamide at concentrationsup to 1,100 mg/ml.

[0033] It is believed that the combination of Ifosfamide, HPBCD andwater also provides stability to the Ifosfamide compositions of thepresent invention.

[0034] It is well known that the treatment with Ifosfamide is associatedwith serious side effects such as haemorrhagic cystitis. To counteractthese side effects, Mesna is practically always administered withIfosfamide injections. However, there are other side effects such asmyelosuppression, cardiac arrythmias, CNS disturbances, nephrotoxicity,haematological and gastro-intestinal reactions which are not addressedby the co-administration of Mesna nor by the Ifosfamide compositionscurrently available.

[0035] Thus, the toxicity of the aqueous compositions of the presentinvention were evaluated against a conventional marketed Ifosfamideproduct (Holoxan™). The compositions of present invention when studiedin Swiss albino mice showed lesser toxic effects such as convulsions,myelosuppressions, and hepatotoxicity. The mortality rate was found tobe significantly less in animals treated with the compositions of thepresent invention compared to a conventional marketed product. LD₅₀values demonstrate the reduced toxic nature of the compositions of thepresent invention. Such data is provided below in the Examples andTables.

[0036] Not being bound by theory, it is believed that Ifosfamide, wholeor in part, is complexed in the HPBCD cavity in an aqueous solution. Onsystemic administration, it is believed that the drug is released fromthe cavity in to the blood stream. The free drug and the drug-HPBCDcomplex will be in equilibrium and the metabolism of the free Ifosfamideshifts the equilibrium resulting in the release of free drug. Presenceof Ifosfamide in complex form and optimum levels of free drug in theblood may prevent the interaction of Ifosfamide with healthy tissues andorgans thereby preventing undesirable side effects.

[0037] In the present invention, Ifosfamide and HPBCD may form asynergistic combination that provides the reduced toxicity both inpresence and absence of Mesna as shown by LD₅₀ values in mice andhaemorrhagic cystitis studies in rats. The Ifosfamide compositions ofthe present invention having reduced toxicity are of advantage in thetreatment of a variety of solid tumours including those of the cervix,endometrium, lung, ovary, testes and thymus as well as in sarcoma and inthe treatment of Burkitts lymphoma.

[0038] The solubility of Ifosfamide in water is about 100 mg/ml. Thepresent invention makes it possible to obtain an aqueous compositioncontaining Ifosfamide in a concentration up to 1,100 mg/ml. Concentratedsolutions offer additional advantages such as safety by virtue of lesshandling, and thus less exposure of clinicians to cytotoxic Ifosfamideduring administration. Further, decreased handling provides an increasein the assurance of sterility.

[0039] Accordingly, in the present invention, in the aqueouscompositions, Ifosfamide may be present at a concentration up to 1,100mg/ml. Preferably the Ifosfamide is present at a concentration of 1-200mg/ml, preferably at 10-100, preferably at 40-50 mg/ml, more preferablyat 200-500 mg/ml and most preferably at 500-1,100 mg/ml.

[0040] Hydroxy propyl-β-cyclodextrin (“HPBCD”) is a partiallysubstituted poly(hydroxypropyl)ether of beta cyclodextrin. Thehydroxypropyl groups are randomly substituted onto hydroxyl groups ofthe cyclodextrin and the amount of substitution is reported as averagedegree of substitution or number of hydroxypropyl groups percyclodextrin. Alternatively, amount of substitution is reported as molarsubstitution (MS) or the average number of substitution peranhydroglucose unit in the ring of the cyclodextrin. Molar substitutioncan have an effect on the binding of guest molecules to HPBCD. At a lowdegree of substitution, binding is very similar to that of theunmodified beta-cyclodextrin. Increasing the molar substitution can leadto a weakened binding due to steric hindrance. HPBCD having molarsubstitution between 0.05 to about 2 is preferable. HPBCD having molarsubstitution between 0.3 to about 1.5 is preferred. HPBCD having molarsubstitution between 0.5 to about 1.2 is more preferred.

[0041] HPBCD may be present in the aqueous composition at a molar ratioof Ifosfamide to HPBCD at 100:0.1 to 1:300; preferably 100:0.25 to1:100; more preferably 100:1 to 1:20. Other preferred molar ratio ofIfosfamide to HPBCD include 100:3.3 to 1:2.5. No significant changes inthe toxicity profile were observed for compositions containingIfosfamide:HPBCD in the ratios of 1:6 to 10:0.25.

[0042] In manufacturing concentrated aqueous Ifosfamide compositions ofthe present invention, the amount of HPBCD required varies with theconcentration of Ifosfamide. Initially, as the concentration ofIfosfamide is increased, so too does the requirement for HPBCD increase.However, at certain point the requirement for HPBCD becomes less. At thehigher end of Ifosfamide concentrations, HPBCD and water content islimited due to volume constraints. Therefore the ratio of Ifosfamide toHPBCD should be chosen, based on the teachings present herein, to allowfor the manufacture of the concentrated Ifosfamide compositions of thepresent invention.

[0043] The pH of aqueous compositions of the present invention withoutany additives may be usually between 3.0-9.0. A preferably pH for thecompositions of the present invention is between 5.0-8.0, but the pH maydrop upon storage. The stabilisation of pH of the composition mayrequire suitable buffers.

[0044] Aqueous compositions according to the present invention may alsoinclude pharmaceutically acceptable additives for the purposes of pHstabilisation, preservation (when the composition is diluted foradministration), isotonicity adjustment, stabilisation againstoxidation, chelating agents, anti-crystallising agents and othersuitable excipients. Some of the pharmaceutically acceptable additivesmay be present in the aqueous solution to which the Ifosfamide and HPBCDare added and/or some of them may be added separately as a solution inwater before making up the volume in the final composition.

[0045] Compositions in accordance with the present invention may,require buffers to adjust and stabilise the pH.

[0046] Suitable buffering agents for compositions of the presentinvention include, but are not limited to, Phosphate buffer, Citratebuffer, Glycine buffer, Histidine buffer containing any of the commonlyused compounds or a mixture of compounds such as Citric acid, Sodiumcitrate, Potassium citrate, Glycine, hydrochloride, Phosphoric acid,Sodium phosphate, Disodium hydrogen phosphate, Sodium dihydrogenphosphate, Potassium phosphate, Dipotassium hydrogen phosphate,Potassium dihydrogen phosphate, Histidine hydrochloride, Sodiumhydroxide, Potassium hydroxide and Hydrochloric acid. Preferably thebuffer used comprises a mixture of Sodium dihydrogen phosphate andDisodium hydrogen phosphate.

[0047] Of the other conventional additives, suitable tonicity agents forcompositions of the present invention include, but are not limited to,glycerin, sodium chloride, maltose, mannitol, dextrose and mixturesthereof.

[0048] Similarly, suitable preservatives for compositions of the presentinvention may include, but are not limited to, methyl hydroxy benzoicacid, propyl hydroxy benzoic acid, phenol, benzyl alcohol and sodiumbenzoate.

[0049] Compositions of the present invention may contain suitablechelating agents such as ethylenediaminetetraacetic acid (EDTA) and itssalts and Desferoximine methane sulfonate (Desferal™).

[0050] Compositions of the present invention may also contain suitableantioxidants such as, ascorbic acid, sodium bisulfite, sodiummetabisulfite, butylated hydroxy anisole and butylated hydroxy toluene.

[0051] Also, the composition of the present may contain substances suchas Glycerin as anti-crystallizing agents.

[0052] The present invention also provides for a process ofmanufacturing aqueous Ifosfamide compositions. The process involvesmixing Ifosfamide and HPBCD and water together. Aqueous solutionscontaining Ifosfamide and HPBCD are brought in intimate contact bystirring. Other methods of bringing the Ifosfamide and HPBCD in intimatecontact include, but are not limited to, mixing, sonicating, heating andhomogenising. Pharmaceutically acceptable additives such as buffers,tonicity agents, preservatives, chelating agents, antioxidants,anti-crystallizing agents as required by parenteral dosage form may bepresent in the aqueous solution to which the Ifosfamide and HPBCD isadded. Alternatively, they can be added separately as a solution inwater before making up the volume.

[0053] In preparing aqueous Ifosfamide compositions, to incorporatelarger amounts of Ifosfamide, HPBCD is dissolved in minimum quantity ofwater and Ifosfamide is solubilized by intimate stirring.Pharmaceutically acceptable additives, if required by parenteral dosageform, are added as such or as solutions into the Ifosfamide-HPBCDsolution. Finally the remaining quantity of water is added to makeup tothe required volume followed by mixing to achieve a homogenous solution.

[0054] The composition may be rendered non-pyrogenic, if required, bypassing through Tangential Flow Filtration System (TFF) beforesterilisation.

[0055] In processes of the present invention the Ifosfamide and HPBCDmay be present in the concentrations and ratios as described above withrespect to the aqueous compositions.

[0056] Process of manufacturing the aqueous Ifosfamide compositions ofthe present invention further comprise sterilization of the composition.The compositions may be sterilized by known and acceptable methods.Preferably the composition is sterilised by filtering through asterilising grade filter. Preferably, the solution is filtered through0.2 μm sterilising grade filters.

[0057] After sterilization, it may be desirable to aseptically place thefiltered solutions into sterile containers such as vials, ampoules, orplastic containers. Preferably, after aseptically placing the filteredsolution into sterile containers, the air in the headspace of thecontainers is purged with an inert gas, such as nitrogen, and then thefilled containers are sealed.

[0058] The following examples serve to illustrate aspects of the presentinvention and are meant in any way to limit the scope of the invention.

EXAMPLES

[0059] Ifosfamide used in these Examples was of parenteral gradecomplying with US Pharmacopoeial specifications. The HPBCD used wasmanufactured by Wacker Chemie having molar substitution peranhydroglucose unit by hydroxy propyl groups between 0.5 to 1.2.Equipment used were of conventional nature and the entire processing wasperformed in an area with a controlled environment. Water used in theseExamples was of parenteral grade complying with “Water for Injection”specifications. All other additives used in these Examples were ofparenteral grade.

Example I Preparation of Ifosfamide 50 mg/ml Composition Containing 20%HPBCD in Water

[0060] The following composition was prepared by the procedure givenbelow i. Ifosfamide 10 gm i. HPBCD 40 gm ii. Water q.s. to 200 ml

[0061] Weighed quantity of HPBCD was dissolved in 150 ml of water.Weighed quantity of Ifosfamide was added and mixed for 3 hours. Thevolume was made up to 200 ml with water and mixed. The resultantsolution was filtered through a 0.2 μm filter and filled aseptically insterile glass vials. The glass vials were closed under asepticconditions with sterile Teflon™ coated rubber bungs and sealed usingflip off seals.

[0062] The composition obtained in this Example was analyzed forIfosfamide content by High Pressure Liquid Chromatography (HPLC) methodand was found to contain 51.73 mg/ml of Ifosfamide. The composition hada pH of 6.5.

Example II Stability of Composition of Example I

[0063] The composition obtained in Example I was subjected to long termStability studies at 2-8° C. The stability data at the end of 24 monthsis shown in Table 1. TABLE 1 Stability data of the composition ofExample I Initial % Ifosfamide HPBCD Ifosfamide 24 months at 2-8° C.mg/ml % Buffer content % Ifosfamide content 50 20% — 103.46 99.12

[0064] The above data shows insignificant drop in Ifosfamide contentindicating a good stability.

Example III Toxicity Study of Composition of Example I

[0065] The composition obtained in Example I was subjected to acutetoxicity studies in mice. Experimental details are as follows.

[0066] Animals used: Swiss albino mice of either sex

[0067] Weight range of animals: 20-22 gm

[0068] Number of groups: 10

[0069] Number of animals per group: 10

[0070] Acclimatization: One week under test conditions under controlledtemperature and humidity Test Materials Ifosfamide Injection IdentityComposition of Example I Description Clear colorless solution Route ofadministration Intravenous Comparative material Holoxan ™(reconstituted) Identity Ifosfamide injection U.S.P. Lot No. G 220Manufacturing Date October 2001 Expiry Date September 2003 DescriptionDry powder for reconstitution with water for injection Strength 40 mg/mlon reconstitution Manufacturer German Remedies Limited Route ofadministration Intravenous

[0071] Both the drug solutions were suitably diluted with 5% DextroseInjection and administered intravenously. Ifosfamide in the doses of 400mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg and 800 mg/kg body weight wasadministered in 10 different groups of animals, each group consisting of10 animals.

[0072] The animals were kept under observation for 14 days and mortalityrecorded at the end of 7 days. The LD₅₀ dose (i.e. the dose that islethal to 50% of animals) is shown in Table 2. TABLE 2 LD₅₀ dose ofComposition of Example I and Holoxan ™ Composition LD₅₀ (mg/kg Bodyweight) Example I 648.04 Holoxan ™ 562.16

[0073] The above data clearly indicates that composition of Example I isless toxic compared to the Conventional formulation.

Example IV Preparation of Ifosfamide Composition Containing 10% HPBCD inPhosphate Buffer

[0074] The following composition was prepared by the procedure givenbelow i. Ifosfamide   10 gm ii. HPBCD   20 gm iii. Disodium hydrogenphosphate  0.1 gm iv. Sodium dihydrogen phosphate 0.06 gm v. Water q.s.to 200 ml

[0075] Weighed quantities of Disodium hydrogen phosphate and Sodiumdihydrogen phosphate were dissolved in 160 ml of water. Weighed quantityof HPBCD was added and dissolved slowly under stirring in this buffersolution. Weighed quantity of Ifosfamide was gradually added understirring to the buffered HPBCD solution and mixed for 3 hours. Thevolume was made up to 200 ml with water and mixed. The resultantsolution was filtered through a 0.2 μm filter and filled aseptically insterile glass vials. The air in the headspace of the vials was purgedwith nitrogen and the glass vials were closed under aseptic conditionswith sterile Teflon™ coated rubber bungs and sealed using flip offseals.

[0076] The composition obtained in this Example was analyzed forIfosfamide content by High Pressure Liquid Chromatography (HPLC) methodand was found to contain 50.23 mg/ml of Ifosfamide. The composition hada pH of 7.2.

Example V Preparation of Ifosfamide 50 mg/ml Composition in PhosphateBuffer

[0077] To ascertain the toxicity reducing nature of HPBCD by toxicitystudies a comparative composition containing Ifosfamide in buffer(without HPBCD) was prepared by the procedure given below. i. Ifosfamide  10 gm ii. Disodium hydrogen phosphate  0.1 gm iii. Sodium dihydrogenphosphate 0.06 gm iv. Water q.s. to 200 ml

[0078] Weighed quantities of Disodium hydrogen phosphate and Sodiumdihydrogen phosphate were dissolved in 180 ml of water. Weighed quantityof Ifosfamide was gradually added under stirring to the buffer solutionand mixed for 3 hours. The volume was made up to 200 ml with water andmixed. The resultant solution was filtered through a 0.2 μm filter andfilled aseptically in sterile glass vials. The air in the headspace ofthe vials was purged with nitrogen gas and the glass vials were closedunder aseptic conditions with sterile Teflon™ coated rubber bungs andsealed using flip off seals.

[0079] The composition obtained in this Example was analyzed forIfosfamide content by High Pressure Liquid Chromatography (HPLC) methodand was found to contain 50.1 mg/ml of Ifosfamide. The composition had apH of 6.5

Example VI Acute Toxicity Study of the Compositions Prepared in ExampleIV and Example V

[0080] Animals used: Swiss albino mice of either sex

[0081] Weight range of animals: 20-22 gm

[0082] Number of groups: 25

[0083] Number of animals per sub group: 8

[0084] Acclimatization: One week under test conditions under controlledtemperature and humidity Test Materials Ifosfamide Injection IdentityComposition of Example IV Description Clear colorless solution Route ofadministration Intravenous Comparative material 1. Ifosfamide InjectionIdentity Composition of Example V Description Clear colorless solutionRoute of administration Intravenous Comparative material 2. Holoxan ™(reconstituted) Uroprotective material Uromitexan ™ Identity MesnaInjection Lot No. G 168 Manufacturing Date October 2001 Expiry DateSeptember 2004 Description Clear & colorless solution for IntravenousInjection Strength 100 mg/ml Manufacturer German Remedies Limited Routeof administration Intravenous

[0085] Experimental details are as follows.

[0086] The compositions obtained in Example IV and Example V weresubjected to acute toxicity studies in mice. A conventional formulation,Holoxan™ was reconstituted as directed by the manufacturer and was usedas a control. The doses of Ifosfamide selected for the study were 400mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg and 800 mg/kg body weight. TheIfosfamide solutions of compositions of Example IV, Example V andHoloxan™ were suitably diluted with 5% Dextrose Injection as such andalso with Mesna at a dose of 20% of Ifosfamide dose and administeredintravenously in 25 different groups of animals, each group consistingof eight animals.

[0087] The animals were kept under observation for 14 days and mortalityrecorded at the end of 7 days. The LD₅₀ values of compositions ofExample IV, Example V, and Holoxan™ are shown in Table 3. TABLE 3 TheLD₅₀ values of compositions of Example IV, Example V and Holoxan ™ HPBCDMesna Dose as concen- % of Ifosfamide LD₅₀ (mg/kg Composition trationBuffer Dose Body weight) Example IV 10% phosphate — 661.57 Holoxan ™ nilnil — 562.16 Example V nil phosphate — 566.00 Example IV 10% phosphate20 669.54 Holoxan ™ nil nil 20 580.00

[0088] The above data clearly indicates that composition of Example IVis less toxic, both in presence and absence of Mesna, compared to theconventional formulation, as well as Ifosfamide in buffered solution.

Example VII Repeat Dose Toxicity Studies of Composition of Example IV

[0089] The composition obtained in Example IV along with conventionalformulation Holoxan™ were subjected to repeat dose toxicity study inmice to evaluate the effect of Ifosfamide compositions on haematologicaland biochemical parameters. Experimental details are as follows. Animalsused Swiss albino mice of either sex Weight range of animals 20-22 gmNumber of groups  7 Number of animals per group 10 Acclimatization Oneweek under test conditions under controlled temperature and humidity.Test Materials Composition of Example IV Comparative material Holoxan ™Uroprotective material Uromitexan ™

[0090] The animals were injected Ifosfamide along with Mesna (as 20% ofIfosfamide Dose) at daily doses of 80 mg/kg, 100 mg/kg and 120 mg/kg,intravenously for seven days. The untreated group was used as a control.The animals were observed during the study period of 14 days formortality, haematological and biochemical changes. The total WBC countwas done before and after treatment with composition of Example IV andHoloxan™. The values are shown in Table 4. TABLE 4 The total WBC countof animals treated with composition of Example IV and Holoxan ™ TotalWBC (cells/microliter) ± SEM Dose (mg/kg Example IV Holoxan ™ Controlbody weight) Pre Post Pre Post Pre Post Ifosfamide Mesna treatmenttreatment treatment treatment treatment treatment 80 16 6220.00 ± 631.733170.00 ± 478.21 4870.00 ± 310.94 1840.00 ± 131.83 7920.00 ± 539.9310980.0 ± 666.65 100 20 5760.00 ± 435.44 6440.00 ± 1764.2 5980.00 ±806.77 2220.00 ± 237.02 120 24 5900.00 ± 481.91 4420.00 ± 1102.0 8150.00± 904.61 1362.50 ± 301.15

[0091] The animals treated with Example IV and Holoxan™ showed reductionin total WBC count. However, animals treated with Holoxan™ showed severeleucopoenia compared to composition of Example IV. This indicates a lesstoxic nature of composition of Example IV.

[0092] To study the effect of composition of Example IV and Holoxan™ onLiver function, the Serum Glutamate Oxaloacetate Transaminase (SGOT)levels of treated and untreated control group were analysed. The SGOTlevels of treated and untreated control groups are shown in Table 5.TABLE 5 The average SGOT levels of animals treated with composition ofExample IV, Holoxan ™ and untreated control Dose of Ifosfamide Dose ofMesna (mg/kg body (mg/kg body SGOT level (U/L) weight) weight) ExampleIV Holoxan ™ Control 80 16 129.15 158.50 136.4 100 20 147.8 299.65 12024 164.25 309.15

[0093] As per the above data, the elevation of SGOT levels in animalstreated with Example IV was slightly higher than that of control group,whereas in animals treated with Holoxan™ increase in SGOT values washighly significant. This indicates reduced hepatotoxic nature ofcomposition of Example IV.

Example VIII Hemorrhagic Cystitis Studies of Composition IV

[0094] The composition obtained in Example IV along with conventionalformulation Holoxan™ were subjected to Hemorrhagic cystitis studies inrats to evaluate their bladder toxicity. Experimental details are asfollows. Animals used Wistar rats of either sex Weight range of animals100-150 gm Number of groups 9 Number of animals per group 2Acclimatization One week under test conditions under controlledtemperature and humidity. Test Materials Composition of Example IVComparative material. Holoxan ™ Uroprotective material Uromitexan ™

[0095] Study Design

[0096] Animals were divided into 9 groups and each group comprised twoanimals. Ifosfamide alone and with Mesna was administered via theintravenous route at doses of 400 mg/kg and 500 mg/kg bodyweight. Thegroup treated with Dextrose Injection was used as a control.

[0097] The animals were sacrificed 24 hours after injection. The urinarybladders of all the animals were collected and were fixed in 10%formalin for 48 hours. Histopathological slides of the organ wereprepared and subjected to microscopic examination. Table 6 depicts theevaluation results on hemorrhagic cystitis of two formulations ofIfosfamide. TABLE 6 Scoring of hemorrhagic cystitis Score Sl noIfosfamide Dose Mesna Dose Example IV Holoxan ™ 1 400 — 1+ 2+ 2 400 — N3+ 3 500 — 1+ 3+ 4 500 — 1+ 1+ 5 400 80 N 1+ 6 400 80 N 1+ 7 500 100 N1+ 8 500 100 N 2+

[0098] Results: Holoxan moderate to severe hemorrhagic cystitis Holoxanwith Mesna mild to moderate hemorrhagic cystitis Example IV mild tomoderate hemorrhagic cystitis Example IV with Mesna No hemorrhagiccystitis

[0099] The above findings conclusively proved that the composition ofExample IV has less bladder toxicity than the conventional formulationHoloxan™.

Example IX Stability Study of Composition of Example IV

[0100] The composition obtained in Example IV was subjected to stabilitystudies at 2° C.-8° C. The samples at the end of 6 and 12 months wereanalysed by HPLC method. The data is shown in Table 7. TABLE 7 Stabilitydata of composition of Example IV Storage condition Description %Ifosfamide content Initial Clear, colourless liquid 50.2 mg/ml 2° C.-8°C. - 6 M  Clear, colourless liquid 50.9 mg/ml 2° C.-8° C. - 12 M Clear,colourless liquid 49.1 mg/ml

[0101] The above data shows insignificant drop in Ifosfamide content at2° C.-8° C. indicating good stability.

[0102] Numerous compositions of the present invention comprisingdifferent concentrations of Ifosfamide and HPBCD are shown in the Table8. TABLE 8 Other embodiments of the present invention ExampleIngredients X XI XII XIII XIV XV XVI Ifosfamide 10.0 g 10.0 g 10.0 g20.0 g  100 g  100 g 200 g HPBCD 40.0 g 80.0 g 20.0 g 40.0 g 40.0 g 80.0g 10.0 g Disodium  0.1 g  0.1 g —  0.1 g — — — hydrogen phosphate Sodium0.06 g 0.06 g — 0.06 g — — — dihydrogen phosphate Water to make qs to qsto qs to qs to qs to qs to qs to up the volume 200 ml 200 ml 200 ml 200ml 200 ml 200 ml 200 ml Ifosfamide 50 mg/ml 50 mg/ml 50 mg/ml 100 mg/ml500 mg/ml 500 mg/ml 1000 mg/ml concentration

[0103] The compositions shown in Table 8 were prepared by the proceduresgiven below.

Example X

[0104] Ifosfamide (10 g) and HPBCD (40 g), Disodium hydrogen phosphate(0.1 gm) and Sodium hydrogenphosphate (0.06 gm) were taken in avolumetric flask of 200 ml capacity. Water for injection was slowlyadded into it with intermittent mixing to get 200 ml clear homogenoussolution. The resultant solution was passed through sterile 0.2 μmfilter and filled aseptically in sterile 10 ml glass vials. The air inthe headspace was purged with nitrogen gas and the glass vials wereclosed under aseptic conditions with sterile Teflon™ coated rubber bungsand sealed using flip-off seals.

Example XI

[0105] Ifosfamide (10 gm) and HPBCD (80 gm), Disodium hydrogen phosphate(0.1 gm in 10 ml of water) and Sodium hydrogen phosphate (0.06 gm in 10ml of water) were taken in a volumetric flask of 200 ml capacity. Waterfor injection was slowly added into it with intermittent mixing to get200 ml clear homogenous solution. The resultant solution was passedthrough sterile 0.2 μm filter and filled aseptically in sterile 10 mlglass vials. The air in the headspace was purged with nitrogen gas andthe glass vials were closed under aseptic conditions with sterileTeflon™ coated rubber bungs and sealed using flip-off seals.

Example XII

[0106] Weighed quantity of HPBCD was dissolved in 40 ml of water. To theconcentrated solution of HPBCD, Ifosfamide was added gradually and themixture was stirred at a moderate speed for 1 hour. The clear solutionwas then diluted to 200 ml with water. The resultant solution wasfiltered through 0.2 μm filter and filled aseptically in sterile glassvials. The air in the headspace of the vials was purged with nitrogengas and the glass vials were closed under aseptic conditions withsterile Teflon™ coated rubber bungs and sealed using flip off seals.

Example XIII

[0107] The compositions were prepared by following the procedure ofExample IV using the components in the amounts mentioned in Table 8.

Example XIV

[0108] Weighed quantity of HPBCD was dissolved in 80 ml of water. To theconcentrated solution of HPBCD, Ifosfamide was added gradually anddissolved by stirring. The volume was made up to 200 ml with water andmixed. The resultant solution was filtered through 0.2 μm filter andfilled aseptically in sterile glass vials. The air in the headspace ofthe vials was purged with nitrogen gas and the glass vials were closedunder aseptic conditions with sterile Teflon™ coated rubber bungs andsealed using flip off seals. The composition in this example wasanalysed for Ifosfamide content by HPLC as was found to contain 500.3mg/ml of Ifosfamide.

Example XV

[0109] The composition was prepared by following the procedure ofExample XIV using the components in the amounts mentioned in Table 8.The composition in this example was analysed for Ifosfamide content byHPLC as was found to contain 500.28 mg/ml of Ifosfamide.

Example XVI

[0110] Ifosfamide (200 gm) and HPBCD (10 gm) were taken in a volumetricflask of 200 ml capacity. Water for injection was slowly added into itwith intermittent mixing to get a 200 ml clear homogenous solution. Theresultant solution was filtered through 0.2 μm filter and filledaseptically in sterile glass vials. The air in the headspace of thevials was purged with nitrogen gas and the glass vials were closed underaseptic conditions with sterile Teflon™ coated rubber bungs and sealedusing flip off seals. The composition in this example was analysed forIfosfamide content by HPLC as was found to contain 1025.5 mg/ml ofIfosfamide.

We claim:
 1. An aqueous Ifosfamide composition having reduced toxicityfor parenteral administration comprising Ifosfamide and2-hydroxylpropyl-β-cyclodextrin, wherein the Ifosfamide is present at aconcentration up to 1,100 mg/ml.
 2. The composition of claim 1, whereinthe Ifosfamide is present at 1-200 mg/ml.
 3. The composition of claim 1,wherein the Ifosfamide is present at 10-100 mg/ml.
 4. The composition ofclaim 1, wherein the Ifosfamide is present at 40-50 mg/ml.
 5. Thecomposition of claim 1, wherein the Ifosfamide is present at 200-500mg/ml.
 6. The composition of claim 1, wherein the Ifosfamide is presentat 500-1,000 mg/ml.
 7. The composition of claim 1, wherein theconcentration of Ifosfamide is greater than 1,000 mg/ml.
 8. Thecomposition of claim 1, wherein the 2-hydroxypropyl-β-cyclodextrin has amolar substitution by hydroxy propyl groups of 0.05-2.
 9. Thecomposition of claim 1, wherein the 2-hydroxypropyl-β-cyclodextrin has amolar substitution by hydroxy propyl groups of 0.3-1.5.
 10. Thecomposition of claim 1, wherein the 2-hydroxypropyl-β-cyclodextrin has amolar substitution by hydroxy propyl groups of 0.5-1.2.
 11. Thecomposition of claim 1 wherein the molar ratio of Ifosfamide to2-hydroxypropyl-β-cyclodextrin is 100:0.1-1:300.
 12. The composition ofclaim 1, wherein the molar ratio of Ifosfamide to2-hydroxypropyl-β-cyclodextrin is 100:0.25-1:100
 13. The composition ofclaim 1, wherein the molar ratio of Ifosfamide to2-hydroxypropyl-β-cyclodextrin is 100:1-1:20
 14. The composition ofclaim 1, wherein the molar ratio of Ifosfamide to2-hydroxypropyl-β-cyclodextrin is 100:3.3-1:2.5.
 15. The composition ofclaim 1, further comprising pharmaceutically acceptable buffers,tonicity agents, preservatives, chelating agents, antioxidants, oranti-crystallizing agents.
 16. The composition of claim 15, wherein thepH of the composition is between 3.0-9.0.
 17. The composition of claim16, wherein the pH of the composition is between 5.0-8.0.
 18. Thecomposition of claim 15, wherein the buffering agent is selected fromthe group consisting of Sodium dihydrogen phosphate, Disodium hydrogenphosphate. Dipotassium hydrogen phosphate, Potassium dihydrogenphosphate, Histidine HCl, Sodium Hydroxide, Phosphoric acid, andHydrochloric acid and mixtures thereof.
 19. The composition of claim 18,wherein the buffering agent is a mixture of Sodium dihydrogen phosphateand Disodium hydrogen phosphate.
 20. A process for the preparation of anaqueous Ifosfamide composition comprising bringing in intimate contactIfosfamide, 2-hydroxypropyl-β-cyclodextrin, and water to form an aqueousIfosfamide/2-hydroxyl-β-cyclodextrin solution.
 21. The process of claim20, wherein the Ifosfamide is present at a concentration of 1-200 mg/ml.22. The process of claim 20, wherein the Ifosfamide is present at aconcentration of 10-100 mg/ml.
 23. The process of claim 20, wherein theIfosfamide is present at a concentration of 40-50 mg/ml.
 24. The processof claim 20, wherein the Ifosfamide is present at a concentration of200-500 mg/ml.
 25. The process of claim 20, wherein the Ifosfamide ispresent at a concentration of 500-1,000 mg/ml.
 26. The process of claim20, wherein the Ifosfamide is present at a concentration of greater than1,000 mg/ml.
 27. The process of claim 20, wherein the2-hydroxypropyl-β-cyclodextrin has a molar substitution by hydroxypropyl groups of 0.5-1.2.
 28. The process of claim 20, wherein the molarratio of Ifosfamide to 2-hydroxypropyl-β-cyclodextrin is 100:0.1-1:300.29. The process of claim 20, wherein the molar ratio of Ifosfamide to2-hydroxypropyl-β-cyclodextrin is 100:0.25-1:100
 30. The process ofclaim 20, wherein the molar ratio of Ifosfamide to2-hydroxypropyl-β-cyclodextrin is 100:1-1:20
 31. The process of claim20, wherein the molar ratio of Ifosfamide to2-hydroxypropyl-β-cyclodextrin is 100:3.3-1:2.5
 32. The process of claim20, further comprising adding one or more pharmaceutically acceptablebuffers, tonicity agents, preservatives, chelating agents, antioxidants,or anti-crystallizing agents while bringing in intimate contact theIfosfamide with the 2-hydroxylproplyl-β-cyclodextrin.
 33. The process ofclaim 20, further comprising adding one or more pharmaceuticallyacceptable buffers, tonicity agents, preservatives, chelating agents,antioxidants, or anti-crystallizing agents to theIfosfamide/2-hydroxylproplyl-β-cyclodextrin solution.
 34. The process ofclaim 32, further comprising sterilization by filtering through asterile 0.2 μm filter.
 35. The process of claim 33, further comprisingaseptically placing the solution into sterile containers followed bypurging of air in the containers with an inert gas, followed by sealingthe containers.
 36. An aqueous Ifosfamide composition according to claim1 wherein each ml of the composition comprises: Ifosfamide: 500 mg; and2-hydroxylpropyl-β-cyclodextrin: 400 mg.
 37. An aqueous Ifosfamidecomposition according to claim 1 wherein each ml of the compositioncomprises: Ifosfamide: 1000 mg; and 2-hydroxylpropyl-β-cyclodextrin: 50mg.
 38. An aqueous Ifosfamide composition according to claim 1 whereineach ml of the composition comprises: Ifosfamide: 50 mg;2-hydroxylpropyl-β-cyclodextrin: 100 mg; Sodium dihydrogen phosphate:0.3 mg; and Disodium hydrogen phosphate: at 0.5 mg.